Dengue epidemics caused by the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical regions. A safe and effective vaccine against dengue is still not available. The current strategy for dengue immunization favors the use of a vaccine containing each of the four serotypes. We previously employed full-length dengue type 4 virus (DEN4) cDNA to construct a viable intertypic dengue virus of type 1 or type 2 antigenic specificity that contained the genes for the capsid-premembrane-envelope (C-PreM-E) structural proteins of DEN1 or PreM-E structural proteins of DEN2 substituting for the corresponding DEN4 genes. Chimeras DEN1/DEN4 and DEN2/DEN4 which express the nonstructural proteins of DEN4 and the C-PreM-E structural proteins of DEN1 or the PreM-E structural proteins of DEN2, and therefore the antigenicity of type 1 or type 2, were used to immunize rhesus monkeys. Monkeys in the control groups were inoculated with parental DEN1, DEN2, or cDNA-derived DEN4. Three of four monkeys immunized with DEN1/DEN4 developed serum neutralizing antibodies against DEN1, and were protected against subsequent DEN1 challenge. All four monkeys immunized with DEN2/DEN4 developed serum neutralizing antibodies against DEN2 and were protected against DEN2 challenge. DEN1- and DEN2- immunized monkeys were protected against homologous virus challenge, but DEN4-immunized animals became viremic on cross- challenge with DEN1 or DEN2. In a second experiment, eight monkeys were immunized with an equal mixture of DEN1/DEN4 and DEN2/DEN4. Each of these monkeys developed serum neutralizing antibodies against both DEN1 and DEN2, and were protected against subsequent challenge with DEN1 or DEN2. Chimeric dengue viruses such as those evaluated in this study could be used to provide their serotype-specific antigens in a tetravalent human vaccine.